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. 2022 Dec 2;186(1):112–130.e20. doi: 10.1016/j.cell.2022.11.030

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© 2022 The Authors

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Figure S2 — SARS-CoV-2 attaches to the cilia during the initial stage of infection, related to Figure 2

(A) ACE2 and TMPRSS2 localize to the motile cilia in ciliated HEN cells. Representative double IF staining of ACE2 and TMPRSS2 in combination with ACTUB in HNEs (left panel) and human nasal tissue (right panel).

(B) SARS-CoV-2 neutralization antibody inhibits attachment of SARS-CoV-2 to cilia. Representative SEM images of SARS-CoV-2 virions attaching to motile cilia after pre-treating HNEs with an unrelated control antibody (left image) or SP neutralizing antibodies (right image) for 2 h before SARS-CoV-2 inoculation with MOI of 0.3 from Donor 7. HNEs were fixed after 6 hpi and observed by SEM Quantification of the average number of virus particles on cilia in infected HNEs. Error bars represent mean ± SD (20–30 cilia were quantified from infected HNEs from Donors 7 and 8, see Table S1).

(C) SARS-CoV-2 RBD binds to the cilia in a ciliary ACE2-dependent manner. ALI-cultured HNEs were labeled by SiR-tubulin, a fluorogenic, cell permeable, and highly specific probe for microtubules and treated with pseudovirion, quantum dot-conjugated SARS-CoV-2 receptor binding domain (QD₅₈₅-RBD) for 6 h. Live-cell images were taken at the indicated time points (seconds) with control (upper image) or soluble ACE2 (down image) using Marianas spinning disk confocal (SDC) microscopy. Quantification of the percentage of HNEs with cilia-attached QD₅₈₅-RBD with control or soluble ACE2 was performed on the right panel. Error bars represent mean ± SD (200–300 cells were quantified from HNEs from Donor 5–8, see Table S1). ^∗p < 0.05, paired, two-tailed Student’s t test.

(D–F) Depletion of cilia doesn’t affect the epithelium development and the expression level of ACE2 and TMPRSS2. (D) Representative IF staining of FOXJ1 and ACTUB in scrambled control (shControl) and CEP83 knockdown (shCEP83) HNEs from Donor 1, see Table S1. (E) Representative IF staining for MUC5AC (goblet cells) and ACTUB (ciliated HNEs) with phalloidin in shControl and shCEP83 HNEs. Quantified percentages of MUC5AC-positive cells in the right panel. Error bars represent mean ± SD (2,000–3,000 cells were quantified from shCEP83 and shControl HNEs from Donor 1–4, see Table S1). (F) ACE2, TMPRSS2, and CX3CR1 mRNA expression in shControl and shCEP83 HNEs. Data were collected from Donors 1–4, see Table S1.

(G) CX3CR1 localizes to the cilia in HNEs. Representative double IF staining of CX3CR1 and ACTUB in ALI-cultured HNEs (top panel) and normal human nasal tissue (down panel).

(H and I) Depletion of cilia decreases the respiratory syncytial virus (RSV) and human parainfluenza virus (PIV) infection. RSV (H) and PIV (I) infected HNEs with MOI of 0.3 were stained after 24 (left image) and 48 (right image) hpi. Representative IF staining for either RSV fusion protein (H) or PIV fusion glycoprotein (I) in combination with ACTUB and phalloidin in shControl and shCEP83 HNEs. Quantified percentages of RSV fusion protein (H) and PIV fusion glycoprotein (I) positive HNEs are shown on the right panel. Error bars represent mean ± SD (3,000–4,000 cells were quantified from virus-infected shCEP83 and shControl HNEs from Donor 1–4, see Table S1. (B–I) ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, NS represents not significant, paired, two-tailed Student’s t test. Each dot represents one donor. Scale bars represent 500 nm (B), 10 μm (C), and 20 μm (A, C, G, H, and I).